The Effects And Mechanism Of Size And Concentration Of Iron Oxide Nanoparticles On The Differentiation Of Bone Marrow Mesenchymal Stem Cells

When iron oxide nanoparticles?IONPs?are widely used in magnetic resonance imaging?MRI?,a growing number of studies have shown that iron oxide nano-particles labeled bone marrow mesenchymal stem cells?BMSCs?affects their proliferation and differentiation.Based on previous reports of iron oxide nanoparticles affecting the differentiation of BMSCs,this study investigated the effect on cell toxicity and differentiation of BMSCs that co-cultured with different particle sizes and concentrations of magnetic iron oxide nanoparticles??-Fe₂O₃?.The effect of different particle sizes and concentrations of?-Fe₂O₃ on the activity and differentiation of BMSCs was obtained.The changes of cytoskeleton F-actin and the expression of Lamin A of BMSCs that co-cultured with different particle sizes and concentrations of?-Fe₂O₃were further studied.The possible mechanism of?-Fe₂O₃ influencing the differentiation of BMSCs was preliminarily deduced.The main research contents and results are as follows:Primary BMSCs from 6-week-old SD rats were cultured to the third generation and co-cultured with?-Fe2O3 with particle sizes of 10,20,30,and 50 nm and at concentrations of 10,50,and 100?g/ml for 14 days.The morphology and density of the cells were observed by microscopy,and the cell viability was measured by MTT assay to characterize the cytotoxicity of?-Fe₂O₃ to BMSCs.The results showed that in the late culture period,the cell morphology of the low concentration group and the high concentration group had a certain degree of change,mainly in the low-concentration group cells are mostly slender spindle-shaped and have produced crystalline material;some cells in the high concentration group were round shaped and distributed in groups.The cytotoxicity of?-Fe₂O₃ to BMSCs was time-and dose-dependent,i.e.,as the culture time prolonged and the?-Fe₂O₃ concentration increased,the cell activity was lower and the cytotoxicity was greater,but the overall cell survival rate was greater than80%.It is considered that the concentration of?-Fe₂O₃ used in the experiment is less toxic to BMSCs.To investigate whether different particle sizes and concentrations of?-Fe₂O₃ affect the differentiation of BMSCs,colorimetric assay was used to quantitatively detect alkaline phosphatase?ALP?and glycosaminoglycan?GAG?,which are the markers of osteogenic differentiation and chondrogenic differentiation,secreted by BMSCs after co-culture at different time points.In order to further determine the accuracy of above differentiation results,the contents of Runx2,an osteogenic differentiation-specific transcription factor,and Sox9,a chondrocyte differentiation-related protein,were detected by western blot and the related changes were analyzed.The results showed that different particle sizes and concentrations of?-Fe₂O₃ had an effect on the differentiation of BMSCs and showed a certain regularity,that is,low concentration of?-Fe₂O₃promoted osteogenic differentiation of BMSCs,among them 10 nm,10?g/ml and 30nm,10?g/ml were the most obvious,while the high concentration of?-Fe₂O₃ promoted the chondrogenic differentiation of BMSCs,among which 30 nm,100?g/ml and 50 nm,100?g/ml were the most obvious.In order to explore the mechanism of different particle sizes and concentrations of?-Fe₂O₃ affecting the differentiation of BMSCs,the changes of cytoskeleton F-actin were detected by fluorescent staining and the changes of Lamin A expression were also detected by western blot after co-culture.The results showed that after co-cultured for 3days,the F-actin density of BMSCs in the?-Fe₂O₃ treated group was increased compared with the control group,and the density of F-actin in the 30 and 50 nm high concentration group was increased obviously compared with the control group.However,the changes of F-actin after 7 days of co-culture was consistent with the differentiation.At the same time,after 7 days of co-culture,the expression levels of Lamin A in the 30 nm,100?g/ml and 50 nm,100?g/ml groups were slightly higher than the 10 nm,10?g/ml and 30 nm,10?g/ml groups and controls.After 14 days of co-culture,the expression of Lamin A in the 10 nm,10?g/ml and 30 nm,10?g/ml groups was significantly higher than in the control group.However,the expression of Lamin A in 30 nm,100?g/ml and 50 nm,100?g/ml groups was slightly lower than that of the control group.Combining the effect of iron oxide nanoparticles on the differentiation of BMSCs,the reason of?-Fe₂O₃ nanoparticles affecting differentiation of BMSCs maybe maybe nanoparticles influence the cytoskeleton and Lamin A expression of BMSCs.Based on the above findings,iron oxide nanoparticles with different particle sizes and concentrations will affect the differentiation of BMSCs and influence the cytoskeleton F-actin and the expression of Lamin A of BMSCs.This study revealed that different sizes and concentrations of iron oxide nanoparticles affect the regulation of differentiation of BMSCs and preliminarily infer the mechanism.Providing a reference for the study of iron oxide nanoparticles affecting the differentiation of BMSCs,and scientific basis for it can be used more safely and more widely in organizational restoration projects.